Supplementary information:
Parasitoid secretions immobilise ant colonies by provoking
internecine fighting
J. A. Thomas, J. J. Knapp, T. Akino, S. Gerty, S. Wakamura, D. J. Simcox, J. C.
Wardlaw & G. W. Elmes
Fuller details than space permitted concerning the methods and results of the
experiments described in this Brief Communication are given below.
Methods
Material and test protocols Four pupae containing Ichneumon eumerus and 17
Myrmica schencki colonies were collected from the Pyrenees in 1996-99. Immediately
after emergence, I. eumerus imagos and pupal cases were stored individually at –60oC
to await bioassays or analysis. Ichneumon chemicals were extracted by washing parts
of its pupal case in 100 μl hexane for 5 min, followed by 100 μl ethyl acetate for 1
hour, then 100 μl methanol for 1 hour. The same extraction process was followed with
whole wasps immersed in 200 μl solvents. Each washing was decanted into a clean
vial, sealed with aluminium caps in nitrogen, and stored at -60oC. In most bioassays,
a 5 mm2 teflon dummy, similar in size and texture to an empty pupal case, was dosed
with extracted or synthetic compounds, and placed on a clean coverslip (using clean
forceps) 1 cm from the nest entrance of a laboratory colony of M. schencki. The
response of worker ants was recorded, then, after a gap of 2 hours, a control dummy,
washed with the solvent alone, was introduced. All dummies (including controls)
were cleaned in methanol before dosing. The Myrmica colonies had been established
in standard 15 x 27 x 10 cm perspex nest boxes kept at 25.5 oC ± 2.5 oC and fed a
standard diet1.
Bioassay 1 (Table 1, below summarised as red histograms in Fig 1 in printed paper)
This involved 5 laboratory M. schencki nests from different colonies, containing
brood and 23, 38, 32, 15, 15 workers. Due to the violent reaction of ants to I. eumerus,
quantifying worker behaviours and the proportion of a colony that reacted by eye was
inaccurate with larger colonies (15 workers represent the smallest colonies of M.
schencki found in the wild2). Bioassays were carried out to record ant responses for 30
min to the base (where the chemicals had drained) of pupal cases from which I.
eumerus emerged, to the pupal bases after washing in hexane, and to dummies treated
with the hexane extract (ethyl acetate and methanol treatments produced diminished
responses that are not reported here). Due to the small number of I. eumerus
available, ‘active’ dummies were treated with extract at 0.1 pupal case equivalent.
Differences in the percentages of workers in colonies behaving differently towards
sources and controls (Table 1) were assessed using STATXACT3 on the actual
numbers of ants. First, the data were analysed using odds ratio tests as a stratified set
of 2x2 tables with the four individual nests as strata. Exact versions of tests for
homogeneity of odds ratios4 did not detect any significant (p>0.05) differences
between the nests for any of the parameters analysed; therefore, overall differences
between sources and controls could be assessed using STATXACT’s exact
probability tests for the null hypothesis of a common odds ratio of unity (equivalent to
no difference between the control and source percentages in Table 1, below).
Identification and synthesis of chemicals All extracts were analysed using GC-MS.
Z-9-C20al, Z-9-C22al and Z-9-C24al were synthesized from C20OH, C22OH, and C24OH,
respectively, through oxidation by pyridinium chlorochromate, and were purified with
column chromatography on silica gel. Z-9-C20ol, Z-9-C22ol and Z-9-C24ol were
synthesized from 8-bromooctan-1-ol by acetylene coupling reaction with 1-dodecyne,
1-tetracecyne or 1-hexadecyne, respectively, and subsequent catalytic hydrogenation.
These unsaturated alcohols were purified with column chromatography on silver
nitrate-loaded silica gel and confirmed with enhancement to >99.5% purity in respect
to positional and geometric isomerism.
Bioassay 2 (Table 2, below summarised as blue histograms in Fig 1 in printed paper).
We followed a similar protocol to bioassay 1 to test ant responses to each of the 6
main chemicals synthesised after identification by GC-MS. Six new laboratory ant
colonies were established from different source nests, as in bioassay 1 except that
each contained 100 workers and ant responses were video-recorded for 10 min
periods. The synthetic chemicals were diluted in an appropriate solvent at
concentrations based on GC-MS analysis of wasp and pupal extracts, namely: 0.3
μg/μl Z-9-C20ol, 5 μg/μl Z-9-C22ol, 14 μg/μl Z-9-C24ol, 7 μg/μl Z-9-C22al, 20 μg/μl Z9-C24al all in hexane; 0.3 μg/μl Z-9-C20al in methanol. Six replicates of each test were
made for each of the separate 6 wasp chemicals dosed on teflon dummies compared to
controls. Three mixtures of all chemicals were also tested. (i) homogenous: 10 μl of
each chemical was mixed in a scintillation vial then 5 μl of this mixture was dosed on
each dummy. (ii) layered: 1 μl of each chemical was placed sequentially onto the
surface of a dummy using a syringe. (iii) coated: 1 μl of each chemical was placed in
a clean scintillation vial; a Teflon dummy was submerged in the vial before being
presented to ants. The solvent controls consisted of 1 μl methanol for Z-9-C20al; 1 μl
hexane for every other chemical; and 1 μl methanol + 4 μl hexane for mixtures.
Differences from the controls were calculated using Mann-Witney non-parametric
analyses, after first using a Kruskal Wallace test to determine that, for each class of
behaviour, the combination of chemicals dissolved in hexane differed overall from the
relevant controls.
Results
The data from which the responses of ants were summarised in Figure 1 of the published paper are presented in two tables: Table 1 represents
results in Fig 1 (a) Wasp chemicals (red histograms) of Brief Communication; Table 2 represents results in Fig 1 (b) synthesised chemicals (blue
histograms).
Table 1 Response of laboratory ant colonies to (i) pupal cases quitted by Ichneumon eumerus, (ii) hexane extracts from pupal cases; (iii) pupal cases
after washing with hexane
Source of chemicals
timesec to discovery
Source Control P
(i) Pupal case
-
-
-
Behaviour and % of worker ants in colony responding to source
% colony attracted
% antennate source
% attack source
Source Control P
Source Control P
Source Control P
% attack other ants
Source Control P
85 ± 9.6 16± 7.5
63 ± 15.2
0
0.0001
45 ± 2.7
0
0.0001
0.0001
26 ± 2.5 11 ± 6.6
ns
8 ± 1.5
2 ± 1.2
(ii) Extract from pupal
case on dummy
12 ± 2.5 45 ± 8.7 0.0001
68 ± 8.0
35± 10.7 0.01
29 ± 3.9 18 ± 5.1
ns
17 ± 2.2 2 ± 1.4
(iii) Pupal case after
extraction
42 ± 10.3 40 ± 8.9
16 ± 7.5
27± 10.0 0.046
11 ± 6.6
ns
2 ± 1.2
ns
11 ± 5.3
1 ± 0.7
ns
0.003
ns
0
0
ns
Extracts were presented to ants on Teflon dummies resembling pupal case. Controlsi = pupal case after washing. Controlsii, iii = Teflon dummies washed in pure
hexane.
Table 2: Response of Myrmica schencki ants to six chemicals identified in the secretions of the parasitoid Ichneumon eumerus.
Response of ants
Alcohols
C20
Run to source
Investigate source
Threaten & bite
source
Repelled from
source
Threaten & fight
kin ants
Aldehydes
p
C22
p
C24
p
C20
p
C22
p
5.5 0.004
± 0.7
11.3 0.04
± 1.9
3.0
0.03
± 1.4
0.8
± 0.7
8.2
± 2.7
2.3
±1.4
ns
3.0
± 0.2
12.2
± 4.1
2.3
± 0.8
ns
ns
ns
0.8
± 0.4
7.0
± 1.5
1.0
± 0.5
ns
ns
0.8
± 0.4
6.0
± 1.6
0
2.8 0.03
± 0.9
6.0
± 5.2
ns
2.7
± 0.7
0.03
0
ns
3.3
± 0.9
3.0
± 1.3
1.3
± 0.7
ns
2.7
± 1.5
0.04
0.8
± 0.5
ns
1.0
± 0.5
ns
ns
ns
ns
ns
C24
p
Mixture of
chemicals
mix
p
†
Controls
hexane
methanol
hex +
meth
1.3
± 0.3
4.3
± 2.0
0.5
± 0.2
1.5
± 1.0
7.7
± 2.5
0.2
± 0.04
0.2
± 0.2
3.5
± 1.8
0.2
± 0.2
2.7
ns
± 1.4
12.7 0.02
± 1.3
4.2 0.002
± 1.1
1.7h
± 0.9
9.5h
± 4.5
0.7l
± 0.2
0.02
1.3
± 0.7
ns
10.0c 0.0001
± 4.1
0.3
± 0.1
0
0
ns
2.0
± 0.1
0.04
4.3l
± 1.3
0.5
± 0.2
0.3
± 0.3
0.7
± 0.3
ns
ns
ns,
ns
ns
0.02
Values represent the mean number of behavioural events ± se in response to source dummies treated with each chemical (Ci) and to controls treated
with solvent only; all individual chemicals had hexane as solvent, except C20 aldehyde (Z-9-C20al ) which had methanol: the full mixture of chemicals
had an appropriate mix of hexane and methanol as solvent (see methods). P values give significant differences from appropriate controls (n=6). The
highest value only of three methods mixtures of applying the full mixture of chemicals to dummies is given (h=homogenised, l=layered, c=coated). †A
fourth mixture of the five individually active compounds (i.e. all except C20 aldehyde) induced significant (P = 0.01) running by ants towards the
source.
Supplementary references (from ‘methods’)
1. Wardlaw, J. C., Elmes, G. W. & Thomas, J. A. Techniques for studying Maculinea
butterflies: I Rearing Maculinea caterpillars with Myrmica ants in the laboratory. J.
Insect Conserv. 2, 79-84 (1998).
2. Wardlaw, J. C. & Elmes, G. W. Exceptional colony size in Myrmica species
(Hymenoptera: Formicidae). Entomologist 115, 191-196 (1996).
3. Cytel Software Corporation StatXact 4 for Windows (Cambridge, MA, 1998).
4. Breslow, N. E & Day, N. E. The analysis of case-control studies. IARC Scientific
Publications 32 (Lyon, France, 1980)
Acknowledgements. We thank R. T. Clarke for statistical advice, K. Schönrogge and
A. Phillips for comments and NERC (GR3/12662) and EU Framework V (MacMan)
for research grants.
Correspondence should be addressed to J.A.T (e-mail: jat@ceh.ac.uk)